To diagnose whether a patient is suffering from cancer or has a predisposition, cells need to be sampled from a patient and a thorough analysis of the cell sample is required in order to evaluate whether abnormal or aberrant cells are present. Mainly, a pathologist or other skilled medical personnel will base the diagnosis on specific characteristics of the cells in the sample, such as cell morphology, the presence of certain types of cells or proteins and more. These cytological tests are based on a two-dimensional presentation of the cells present in the sample and mostly require the fixation of cells on a substratum and the use of dyes or stainings to visualize specific features of the cells. This is a time consuming and cumbersome work, and requires well-trained specialists. Moreover, as many of the solutions used to fix and stain the cells, this approach will inevitably lead to loss of cell structures and information stored therein. This might thus interfere with the possibility of a reliable interpretation and diagnosis from the sample. Inadequate processing of a sample may lead to an increased number of false negatives diagnoses. For instance, of the over 50 million cervical cytological PAP smears, which are performed in the USA each year, a high false-negative interpretation rate of 20-40% has been described (Williams et al., 1998), frequently leading to fatal consequences. Most of these false negatives are the result of inadequate sample processing.
Since 1990 many advanced technologies focusing on sampling, smear preparation, or screening quality control have been developed and introduced into the practical work to prevent the false negative rate in screening. These commercial devices can be divided into the following categories based on their approaches: (1) for a better slide preparation to reduce sampling error, such as thin-layered liquid based preparation (ThinPrep™, SurePath, Tripath); (2) for reducing workload and screening error, such as autoscreening system (ThinPrep Imaging System, Cytyc, Boxborough, Mass.) and FocalPoint System (Tripath Imaging, Burlington, N.C.); (3) for laboratory quality control, such as rescreening (Papnet); and (4) for quality assurance, such as proficiency test. However, most of these devices are not designed to assist diagnosis by supplying the calculable parameters to eliminate interpretation errors and inter-observer discrepancy. In addition, it is not applicable for general cytological laboratory because of high cost and technical or linguistic gaps. Thus, without a reproducible and quantitative tool, it is still an unsolved problem for a routine cytological laboratory to improve the diagnostic divergence caused by visual observation.
Therefore, the field of cancer diagnosis is in need for methods and devices that analyses cell samples in a non-destructive, non-detrimental and objective manner, or at least provide information of the status of the sample and the cells present prior to its further processing by a specialist. Preferably, the gathered information is obtained by a three-dimensional analysis method in order to perturb the sampled cells to a minimum prior to analysis. Moreover, three-dimensional information will store substantially more cellular data than conventional two-dimensional information. This will undeniably lead to a more reliable diagnosis method as more accurate information will be obtained from the analyzing sample.
U.S. Pat. No. 2,010,006 089 7 discloses a method and device for non-destructive analysis and characterization of a cell sample. The invention makes use of a digital holographic microscope for analyzing certain parameters of a cell and to determine the number of cells in the sample. U.S. Pat. No. 2,010,006 089 7 does not disclose specific parameters to be measured in order to classify a cell as healthy or aberrant. Therefore, the method as disclosed in U.S. Pat. No. 2,010,006 089 7 can be implemented in a diagnostic system, but can serve there merely as an extra tool for gathering information on a sample, and not as the main determining factor whether a sample contains aberrant cells or not.
Choi et al. (2007) from the Massachusetts Institute of Technology (MIT) describes a method based on tomographic phase microscopy to map 3D structures of suspended or substrate-attached cells and to quantify refractive index measurements. By creating overlapping tomograms, a 3D image of a cell could be reconstructed. The authors state that the refractive index data obtained by their described technique can be used to characterize cell sample aberrations. Specific parameters which are suitable to be used in a diagnostic setting are however not disclosed. Moreover, the technique does not generate a real time three-dimensional image, but rather artificially creates a 3D image by superimposing several two-dimensional images captured by the microscope.
Reshetov et al. (2010) present a method to visualize thyroid cancer cells and to study their morphology by atomic force microscopy (AFM). The authors showed a difference in height of the nucleus, height of cytoplasm and ratio thereof of thyroid cancer cells when compared to benign colloidal goiter cells. Disadvantage of the system is the slow scanning speed of the AFM technique, requiring several minutes for one scan. Other disadvantage is the limited area which can be scanned (only micrometer scale, 100×100 μm in X and Y direction, and 10 μm in Z direction) by an AFM as well as the poor image resolution. Moreover, imaging of liquids, for instance cells in solutions, have been proven to be challenging with conventional AFM. These disadvantages make it unlikely that AFM will be widely implemented in oncocytological diagnostic devices.
There remains a need in the art for an improved, non-destructive method for measuring and obtaining specific cellular parameters of cell samples in a three-dimensional manner, which can be used to diagnose the status of the analyzed cell sample. The method should be readily implemented in a cytological screening and diagnosis system and provide a fast, objective and correct analysis of cell samples thereby limiting the requirement of highly trained personnel and man hours which are currently needed to process and analyze each cell sample.